Creep in nitroimidazole inhibitory concentration among the Entamoeba histolytica isolates causing amoebic liver abscess and screening of andrographolide as a repurposing drug

Infections by Entamoeba histolytica (E. histolytica) lead to considerable morbidity and mortality worldwide and treatment is reliant on a single class of drugs, nitroimidazoles. Treatment failures and intermittent reports of relapse from different parts of world indicate towards development of clinical drug resistance. In the present study, susceptibility testing of clinical isolates of E. histolytica was carried against metronidazole and tinidazole. Additionally, anti-amoebic property of active compounds of Andrographis paniculata was also evaluated. Prevalence of metronidazole resistance gene (nim) in patients attending hospital was also done to get comprehensive insight of present situation of drug resistance in E. histolytica. Mean inhibitory concentration 50 (IC50) value of E. histolytica isolates against metronidazole and tinidazole was 20.01 and 16.1 µM respectively. Andrographolide showed minimum mean IC50 value (3.06 µM). Significant percentage inhibition of E. histolytica isolates by andrographolide was seen as compared to metronidazole (p = 0.0495). None of E. histolytica isolates showed presence of nim gene. However, in stool samples from hospital attending population, prevalence of nimE gene was found to be 76.6% (69/90) and 62.2% (56/90) in diarrheal and non-diarrheal samples respectively. Inhibitory concentration of commonly used nitroimidazoles against clinical isolates of E. histolytica are on rise. Percentage inhibition of E. histolytica isolates by andrographolide was significantly higher than control drug metronidazole.


Results
Confirmation of the samples and demographic details. All the 15 liver aspirates included in the study were found to be positive for E. histolytica through nested multipex polymerase chain reaction (PCR) for Entamoeba sp. However, only 2 (13.3%) stool samples of these ALA patients were positive for E. histolytica. As we have previously reported increased recurrence rate in ALA patients thus, the isolation of the trophozoites were carried out from the liver aspirate samples. Figure 1 shows the presence of the trophozoites in the liver aspirate samples. Among the diarrheal samples included in the present study 3 (3.3%) samples were positive for E. histolytica.
The mean age of the ALA patients was 41.06 ± 7.3 years. Majority (13/15, 86.6%) of the participants were males. Considering their residence, 9 (60%) participants were from rural background and 6 (40%) were from urban areas. Out of the total, 5 (33.3%) participants had received higher education (above class 5th), 7 (46.6%) participants had primary education and 3 (20%) were uneducated. Majority (11/15, 73.3%) participants were employed. Five (33.3%) participants were from low and 10 (66.6%) participants were from middle economic status. In the present study, the incidence of ALA was more frequent in middle-aged males with rural background. However, no significant correlation was found between the demographic factors and the incidence of the ALA owing to the inclusion of only the diseased ALA cases (no controls) and the small sample size of the study.
Drug susceptibility testing of E. histolytica clinical isolates. The mean IC50 value of the E. histolytica isolates against the metronidazole and tinidazole was found to be 20.01 µM and 16.1 µM respectively. The mean IC50 values were significantly higher (p = 0.022) for metronidazole as compared to the tinidazole. Figure 2 Scientific Reports | (2023) 13 Through analysis of the digested fragments from both the enzymes, all the samples positive for nim gene were found to be nim E type. The fragments obtained by digestion from Hin1II and Taq1 restriction enzyme has been shown in Fig. 4. The restriction digestion products obtained by Hin1II enzyme showed a single fragment of 441 bp and Taq1 enzyme produced two fragments of 274 bp and 155 bp on agarose gel electrophoresis.
The sequence analysis of the nim gene PCR products confirmed the presence of nim E gene type. A sequence homology of 99% with the nim E gene type was revealed through Basic local alignment search tool (BLAST) analysis from the available database. (Accession number: AM117602.1, https:// www. ncbi. nlm. nih. gov/ nucco re/ AM117 602.1/).

Discussion
Drug susceptibility testing in E. histolytica has been challenging because of the absence of competent screening assay. Additionally, the available methods are exhaustive and have complex procedures besides requiring expertise and parasite culture facilities 30 . Thus, only a handful of studies are available reporting the drug sensitivity of E. histolytica isolates. The most interesting point was that though the findings of the present study were in agreement with the previous reports, yet the mean IC50 values in the present study were higher than the prior studies 10,31 . A study conducted 18 years before in India on the drug sensitivity of the E. histolytica isolates had shown the mean IC50 value of 13.2 µM and 12.4 µM for metronidazole and tinidazole respectively 10 . The increase in the mean IC50 values can be attributed to the emergence of clinical resistance or due to the differences in the culture techniques of the parasite, strains involved or raw materials used. Studies have reported the minimum MIC of 12.5-25 µM for the laboratory passaged E. histolytica strains. Another study described mean IC50 value of 18.47 µM and a cut off value of > 30 µM for resistance in the E. histolytica isolates 32 . However, majority of the studies on the drug sensitivity of E. histolytica are outdated and there is a dearth of recent data against the commonly used anti-amoebic agents. Additionally, the comparison of the available studies regarding the drug sensitivity of E. histolytica particularly becomes difficult because of the use of the different methods for the measurement of the activity of the drugs.
Various studies have reported the sensitivity of the drugs against the E. histolytica isolates in terms of minimum lethal concentration (MLC), MIC, IC50, effective dose 50 (ED50) etc [32][33][34][35] . However, it has been suggested to report the sensitivity of a drug as molar concentration (µM) to standardize the comparison of efficacy of the drugs especially in cases when metronidazole is compared with other nitroimidazoles with significantly higher molecular weight 31 . The clinical isolates included in the present study were maintained in the monoxenic culture as it has been previously reported that presence of bacterial flora along with the amoeba did not significantly interfere with the performance of the test or the sensitivity values 10,36 .
The percentage inhibition of the E. histolytica clinical isolates by marker active compound andrographolide was significantly higher than the control drug metronidazole. Reports have suggested that andrographolide and its analogues possess some novel mechanism of action responsible for their effects 37 . A recent study using andrographolide based nanoparticles has shown that they generate intracellular reactive oxygen species that promote damage to the vital biomolecules including DNA, proteins and lipids and cause membrane leaking leading to the release of the cytoplasmic components and death 38 .  www.nature.com/scientificreports/ In the malarial parasite, andrographolide had shown inhibitory activity towards the ring-stage of the parasite affecting the protein and nucleic acid synthesis. Andrographolide has been designated as the 'Transcription blocker' in the parasite Plasmodium falciparum 39 . In the andrographolide treated filarial parasite the activity of antioxidant enzymes and glutathione-S-transferase (GSH) has been found to be reduced in concentration dependent manner 40 . Additionally, in parasites treated with andrographolide an increase in the activity of NADPH oxidase has been seen which leads to the development of hydrogen peroxide (H 2 O 2 ) and other hazardous reactive oxygen species splitting the mitochondrial membrane organization 40 . Even though andrographolide has been found to be effective as a traditional medicine in dysentery, a detailed study on the exact mode of action of andrographolide in E. histolytica is lacking.
In the present study, the nim gene was not detected in any of the clinical isolates of E. histolytica. However, the present hospital-based study revealed high level prevalence of the nim gene in the diarrheal as well as nondiarrheal stool samples of the patients. In other anaerobic micro-organisms, such as clinical isolates of Bacteroides spp. and Prevotella spp. the carriage rate of 0-2.8% and 0-8% respectively has been reported 17,[41][42][43] . There is no data on the presence of nim genes in E. histolytica clinical isolates for commenting on this study finding. The nim gene associated metronidazole resistance has been known to vary among the diverse geographical regions 44 . In the present study the decreased susceptibility of the clinical isolates of E. histolytica towards commonly used nitroimidazoles does not seem to be associated with the nim genes. Previous studies have suggested that the reduction in sensitivity to metronidazole in the E. histolytica isolates can be because of several factors, some of which are non-enzymatic 45 . The increased expression of the iron super dismutase (Fe-SOD) and peroxiredoxin along with the decreased expression of ferredoxin1 and TrxR constitutes important component involved in the mechanism of metronidazole resistance in E. histolytica 46 .
High prevalence of nim gene in stool samples of the patients attending the tertiary care centre is in concordance with another study from India that showed high copy number of the nim gene in the stool samples of the patients with gastrointestinal discomfort as well as in the healthy individual from the community 47 . Such high prevalence of the nim gene in this geographical region can be attributed to the OTC sales of the nitroimidazoles (especially metronidazole), which has been linked with the increased frequency of the nim gene induction 48 . Also, the presence of nim gene was found to be significantly associated with the diarrheal stool samples (p = 0.036). This could be due to the significant abundance of the nim gene carrying micro-organisms such as Bacteroides spp. and Prevotella spp. in the diarrheal stool samples 49,50 .
The presence of nim E gene type in all the positive samples has been reported in the present study. Our finding is in-line with the previous studies reported from India which described nim E to be the most common nim gene type circulating in this geographical region 5,47,51 . In contrast to our results, studies from other part of the world have shown nim A gene followed by nim B and nim D gene to be most prevalent type 14,15 . However, no possible correlation between the type of nim gene and the degree of resistance has been ever described in the literature.
The scope of the present work was to analyse the susceptibility pattern of the clinical isolates of E. histolytica towards the commonly used drugs. Along with it, the anti-amoebic property of the marker compounds of A. paniculata was studied. To the best of our knowledge, this is the first report of the anti-amoebic activity of the active compounds from the extracts of leaves of A. paniculata against the clinical isolates of E. histolytica. A detailed study of the toxicity profile, dose determination and the delivery options of the active compounds is required to establish them as a potent anti-amoebic agent in the future.
The study was not without any limitation as only 15 clinical isolates of E. histolytica were included in the study owing to the difficulty in the establishment and maintenance of the parasitic culture. The drug susceptibility assays could not be carried out in the axenic culture due to lack of the culture and maintenance facility. Additionally, the drug susceptibility assay was performed using only two drugs, metronidazole and tinidazole. However, these drugs are the main line of treatment for the E. histolytica infections including ALA cases and therefore provides important insight into the situation.

Material and methods
Ethics statement. The study was ethically approved by Institute Ethical Committee, Faculty of Medicine, Institute of Medical Sciences, Banaras Hindu University (Dean/2016-17/EC/045). All experiments in the present study were performed in accordance with guidelines and regulations provided by the Institute Ethical Committee. The study included only adults and written informed consent was obtained from all the subjects who participated after explaining them the purpose of the study.
Inclusion/exclusion criteria. Patients with well-defined liver abscesses greater than 5 cm diameter which were confirmed through abdominal ultrasound were included in the present study. Patients who have gone through aspiration previously or were on any medication from past four weeks were excluded from the study. Furthermore, the liver aspirate showing presence of associated bacteria either by aerobic culture or by molecular detection through 16-S rRNA primers were excluded from the study 52 .
Clinical samples and isolates of E. histolytica. Fifteen isolates from 15 E. histolytica positive ALA samples from patients attending the Gastroenterology and Radiology department of Sir Sunderlal Hospital, Varanasi, India were included in this study. As the trophozoites are majorly attached to the wall of the abscess, the use of a series of smaller bottles for the collection of the liver aspirate was done to keep the last fraction undiluted by the main mass of material 53 .
The stool samples from these ALA patients were also collected. A pre-tested questionnaire about the demographic details including age, gender, residence, education, employment and economic status of the patients were collected through experienced research scholars. www.nature.com/scientificreports/ Additionally, diarrheal (n = 90) and non-diarrheal (n = 90) stool samples from patients attending different outpatient departments of Sir Sundarlal Hospital, Varanasi were also included to detect the frequency of the nim genes in this geographical region. All the diarrheal samples were inoculated on the Mac Conkey agar medium and blood agar medium for overnight incubation at 37 °C. Additionally screening of common anaerobes from previous literature was done in the diarrheal samples using conventional PCR 29 . Confirmation of the samples. Direct microscopy was performed for all the samples through wet mount to screen for the presence of trophozoites of Entamoeba spp. Nested multiplex PCR was performed for the molecular detection of E. histolytica in all the samples using species specific primers targeting 16S like rRNA gene 54 . The reaction mixture preparation and reaction conditions were as described previously 29 . Isolation and culture of the trophozoites. Trophozoites were isolated from liver aspirate of ALA patients by culture in modified Boeck and Drbohlav's monoxenic medium with few modifications 53,55 . Lockeegg medium was prepared. For this, Locke's solution was prepared by dissolving the 8.0 g sodium chloride (Sigma-Aldrich Chemicals Pvt. Ltd, India), 0. For egg slant preparation, fresh hens' eggs were sterilized by flaming in 70% ethanol and broken into a graduated cylinder. Locke's solution (12.5 ml) per 45 ml of egg was added and then emulsified in a Waring-type blender and filtered through gauze into a flask. A total of 5 ml of the emulsified egg were added to standard culture tubes (16 by 125 mm) and sterilized by inspissation. After cooling of the slants, they were overlayed with 6 ml of Locke's solution.
Deactivated bovine serum (Biological industries, Kibbutz Beit-Haemek, Israel) and sterilized rice starch (VWR International, Poole, United Kingdom) were added to the medium before addition of the samples 56 . Along with it, a loopful of pure colonies of Klebsiella pneumoniae which was susceptible to the metronidazole was inoculated to the culture tubes before the addition of the clinical sample. The sub-culturing was performed every 48 h and the trophozoites were further subjected to drug susceptibility testing mostly after two passages.

Drug susceptibility testing (DST).
For the susceptibility testing, Robinson medium for Entamoeba twin pack (HiMedia laboratories Pvt. Ltd, Mumbai, India) was used. Susceptibility testing to the standard drugs and the active compounds was performed as described previously 10 . Briefly, the trophozoites were harvested from 24 h old culture from the interface of the locke's solution and the egg slant and the count were adjusted to 1 × 10 5 trophozoites per ml. The in-vitro susceptibility testing was performed in 96 wells microtiter plates. The doubling dilutions of the drugs and active compounds was performed to get the required concentrations. Diluted trophozoite suspension was added and the plates were incubated for 4 h at 37 °C. Following incubation, the plate's content was discarded and the plate was washed. Further, 100 µl of nitroblue tetrazolium (HiMedia laboratories Pvt. Ltd, Mumbai, India) was added to each well and the plates were incubated at 37 °C for 45 min. After incubation, the plates were again washed and DMSO (200 µl/well) was added. Thereafter the plates were incubated at 37 °C for 10 min. Following incubation, the optical density (OD) was measured. Each isolate was tested against the drugs and active compounds in duplicates. Each test included a control well containing only media and the trophozoites without drugs and a blank well containing only media. The measurement of OD in each test was done at 540 nm using microtiter plate reader (LisaScan® EM Elisa plate reader, Transasia Bio-Medicals Ltd, India). The percentage of non-viable trophozoites at each concentration was calculated using the formula: The mean IC 50 values for all the clinical isolates against the drugs (metronidazole, tinidazole) and the active compounds (andrographolide, neoandrographolide and andrograpanin) was calculated using "Quest Graph™ IC50 Calculator" (AAT Bioquest, Inc.) Screening of the nim gene. The 15 E. histolytica isolates and stool samples from the diarrheal and nondiarrheal patients were screened for the presence of nim genes. DNA extraction was carried out from all the isolates and the stool samples by using QIAGEN stool mini kit (Qiagen, Germany) as per the manufacturer's instructions.
The detection of nim genes in the E. histolytica isolates and stool samples was carried out by conventional PCR using the universal set of primers, Nim-3 and Nim-5, for all known nim genes 57 .

Percentage of non-viable trophozoites =
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